The family of naturally occurring oligopeptide antibiotics includes amidomycin, anthelvencins A and B, distamycin, kikumycins A and B, netropsin and norformycin. The best characterized members of this family, distamycin and netropsin, exhibit antibiotic, antiviral and antitumor activity. However, the clinical use of these agents has been limited by their high cytotoxicity.
Netropsin and distamycin interact with DNA in a sequence-specific and groove-selective manner by binding to specific nucleotide sequences [(AT).sub.4 and (AT).sub.5, respectively] within the minor groove of B-DNA (Hahn [1975] in Antibiotics III. Mechanism of Action of Antimicrobial and Antitumor Agents, Corcoran and Hahn, eds., Springer Verlag, New York, 79). The firm and sequence specific binding of netropsin is the net result of specific hydrogen bonding, electrostatic attraction and van der Waals interactions (Kopka et al. [1985]Proc. Natl. Acad. Sci. U.S.A. 82, 1376 "Kopka et al.").
Lown et al. (1986) Biochemistry 25, 7408, report the characterization of derivatives of netropsin in which each of the pyrrole units is successively replaced by an imidazole moiety, as well as di- and triimidazole-containing derivatives. The synthesis of the compounds is reported in Krowicki et al. (1987) J. Org. Chem. 52, 3493. DNA binding studies revealed a gradual change in base preference upon replacing pyrrole by imidazole, indicating that the molecular recognition process is complex. In a related study, high field .sup.1 H--NMR was used to compare the sequence specific binding of netropsin and the bis-imidazole analog to a the decadeoxyribonucleotide d-[CGCAATTGCG].sub.2 (Lee et al. [1988]J. Biomol. Struct Dyn. 5, 939). Drug-induced chemical shift changes suggest that the bisimidazole moiety of the analog can accept GC sites, although it binds primarily at AATT. In an analogous study with a netropsin derivative in which one pyrrole moiety was substituted by imidazole, the drug appeared to bind at the AATT site in the minor groove of the DNA (Lee et al. (1988) Biochemistry 27, 445). Quantum chemical studies indicate that the monosubstituted derivative is capable of binding to (GC).sub.n sequences, but the binding energy for (GC).sub.n is reduced relative to (AT).sub.n by approximately one half. Furthermore, a comprehensive circular dichroism study (Burckhardt et al. [1989]Biochim. Biophys. Acta 1009, 11) demonstrates that while imidazole substituted netropsin analogs show decreased binding preference for AT pairs, a real specificity for GC sequences is not observed. Lown et al. (1986) J. Med. Chem. 29, 1210, report that the mono- and di-imidazole substituted netropsin analogs lack cytostatic activity against various tumor cell lines, and the analog with three imidazole moieties has moderate cytostatic activity which is less than that of distamycin. Antiviral activity of the analogs is specific for vaccinia virus and is less than that of the parent compounds.
Kissinger et al. (1987) Biochemistry 26, 5590 disclose the synthesis of a series of netropsin derivatives in which the guanidinium group of the amino terminus of netropsin is replaced with an N-formyl group, and none, one or both of the N-methylpyrrole groups are replaced by N-methylimidazole. The replacement of the guanidinium group with a formyl group renders the compounds monocationic rather than dicationic. DNAase 1 footprinting studies suggest that the bis(imidazole) analog binds to the sequence 5'-CCGT-3' or 5'-ACGG-3'. The contribution of the substitution of the guanidinium group with a formyl group is not clear, since the monocationic analog possessing two N-methylpyrrole groups exhibited binding that was very similar to the binding of netrosin. The biological activity of the analogs was not assessed. The specific binding of the monocationic bis(imidazole) analog to the base sequence 5'-CCGT3' was confirmed by .sup.1 H--NMR studies (Lee et al. [1988]J. Biomol. Struct. Dyn. 5, 1059).
Less et al. (1988) J. Amer. Chem. Soc. 110, 3641, disclose the synthesis of monocationic netrospin analogs with one or two methylene groups at the C-terminus. The analog with one methylene group exhibited preferential binding for the sequence ATTG on the decadeosyribonucleotide [CGCAATTGCG].sub.2, while the analog with two methylene groups exhibited a preference for the sequence AATT, as determined by chemical shift changes.
Lown et al. (1989) J. Med. Chem. 32, 2368 and U.S. Pat. No. 4,912,199 to Lown et al. discloses oligopeptides structurally related to distamycin and netrospin in which the heterocyclic moieties are linked by polymethylene bridges or dicarboxylic acid derivatives, respectively. Enhanced antitumor activity against certain cell types and antiviral activity specific for vaccinia virus as a result of introduction of polymethylene linkers ([CH.sub.2 ].sub.n with n=1,2 and 6-8) was attributed to increased lipophilicity promoting cellular uptake, since DNA binding is comparable to that of the parent compounds.
U.S. Pat. No. 4,738,980 to Arcamone et al. ("Arcamone et al. I") and Arcamone et al. (1989) J. Med. Chem. 32, 774 ("Arcamone et al. II") disclose pyrrole-containing oligopeptide derivatives containing an alkylating group at the N-terminus. In Arcamone et al. II, derivatives containing an N,N-bis(2-chloroethyl) group displayed in vitro cytotoxicity against certain tumor cell lines and in vivo antitumor activity against murine L1210 leukemia
The present invention provides imidazole-substituted oligopeptide derivatives that are conjugated to DNA alkylating agents. The compounds of the present invention are useful as antitumor agents.